CN111454933B - D-carbamoyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acid - Google Patents

Abstract

The invention discloses a D-carbamyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acid, belonging to the technical field of genetic engineering. The mutation of the D-carbamyl hydrolase has higher activity to a plurality of N-carbamyl amino acids, and can catalyze a plurality of aliphatic or aryl substituted amino acid substrates, especially D-N-carbamyl amino acid substrates with larger steric hindrance. The mutants are used for the validation that 500mM L-indolylmethylhydantoin is catalyzed by a hydantoinase process cascade reaction, wherein the yield of a product of the five mutants A200N/R211G/D187N/S207A/E138W in 12 hours of reaction reaches 81.7 percent, compared with WT (40.1 percent), the yield of the product is improved by 2 times, and the reaction time is greatly shortened and only needs 12 hours. The D-carbamyl hydrolase mutant obtained by the invention is particularly suitable for preparing optically pure D-amino acid by a hydantoinase process cascade reaction, and has good industrial application prospect.

Description

D-carbamoyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acid

Technical Field

The invention relates to a D-carbamyl hydrolase mutant and application thereof in synthesis of D-aromatic amino acids, belonging to the technical field of genetic engineering.

Background

D-amino acid, an unnatural amino acid, is commonly used in the synthesis of various pharmaceutical intermediates, e.g., D-p-hydroxyphenylglycine is commonly used as a precursor in the synthesis of cephalosporins (Syldatk C., Advances in Biochemical Engineering Biotechnology,1990, 29-75); d-tryptophan (D-Trp) can be used for synthesizing Octreotide and tadalafil Cialis, which are important drugs for treating acromegaly and erectile dysfunction; D-Trp can also be used for synthesizing peptide drugs for treating dermatitis, comprising: tyrocidinesic (D-Phe-Pro-Trp-D-Trp-Asn-Gln-Tyr-Val-Orn-Leu) and Thymodepressin (γ -D-Glu-D-Trp), etc. (martinez-rodi guez et al, chem. biolivers, 2010,7, 1531-.

D-N-carbamoylase belongs to the 6 th class of nitrilase superfamily, which can hydrolyze D-N-carbamoylamino acid to obtain D-amino acid, and is commonly used to prepare optically pure D-amino acid by cascade reaction with hydantoin racemase and hydantoin enzyme.

In recent years, D-tryptophan and D-p-hydroxyphenylglycine have been studied mainly for the synthesis of D-aromatic amino acids, and for example, Yamamoto et al, 1998, selectively hydrolyzes D-tryptophanyl amine using D-N-amidase and then obtains D-Trp by chemical hydrolysis (EP0853128A 1). Similar to the amidase method, Greenstein, et al, 1957 prepared D-Trp using L-aminoacylase, where only the L-enantiomer of N-acetyl-DL-tryptamine was hydrolyzed and the remaining enantiomer was chemically deacetylated to form D-Trp (Greenstein, J.P., Methods In Enzymology,1957,3: 554-. In 1995, Yamamoto H et al reported that D-tryptophanase could degrade L-Trp from DL-Trp racemate to produce D-Trp, but pyruvate and indole, by-products of this reaction, inhibited tryptophanase activity and thus required removal (Kawasaki et al bioscience Biotechnology and Biochemistry,1995,59: 1938-. The methods reported at present have the defects of 50 percent of theoretical yield, complex process, low yield, enantioselectivity and the like. Although in 1949, Eadie G et al reported that optically pure D-Trp can be prepared by a method in which D-amino acid transaminase is used to react the substrates indolypyruvic acid and D-alanine, the theoretical yield was 100%, but the activity was low, and the final yield was only 13% (Eadie G et al. journal of Biological Chemistry,1949,181: 449-458). The hydantoinase process is a three-enzyme cascade reaction consisting of hydantoin racemase, hydantoinase and carbamoyl hydrolase, and has higher yield because the traditional limitation of 50% conversion rate can be broken through, so that the method is always considered as a method for preparing optically pure D-amino acid efficiently and economically. In the previous research, the inventor has screened a D-carbamoylase derived from Arthrobacter crystallopoietum, and the patent number (201711097767.0) discloses that the D-carbamoylase can catalyze 300mM L-indolylhydantoin to generate corresponding D-tryptophan basically and completely, the enzyme is used for cascade reaction, although the conversion of 300mM substrate can be realized, a large amount of enzyme (50kU/L AaHyuA,25kU/L AtHyuH and 50kU/LAcHyuC) is required to be added, and in the previous research, the D-carbamoylase is found to have enzyme activity obviously lower than that of the previous two steps of reaction, when the substrate concentration is continuously increased, a large amount of D-carbamoylase is required to be further added to complete the conversion, so that the original economy of the process is lost, therefore, in the previous research, the subject group obtains a D-carbamoylase derived from Nitinol indonesia reducer India C115 by a gene mining method, named NiHyuC, the enzyme can realize complete conversion of 1M L-indolylhydantoine, but the problems of low D-carbamyl hydrolase and high enzyme loading (50kU/L AaHyuA,25kU/L AtHyuH and 50kU/L NiHyuC) also exist.

Disclosure of Invention

In order to solve the problems, the invention improves the catalytic activity of the D-carbamyl hydrolase by a protein engineering method, thereby reducing the addition amount of the enzyme, which is very important for the efficient preparation of the D-amino acid by applying a hydantoinase process.

The first purpose of the invention is to provide a D-carbamoyl hydrolase mutant, which is obtained by mutating one or more of glutamic acid at position 138, aspartic acid at position 187, alanine at position 200, serine at position 207 and arginine at position 211 of D-carbamoyl hydrolase with the amino acid sequence shown in SEQ ID NO. 2.

Furthermore, the mutant is obtained by mutating the glutamic acid at the 138 th site of the D-carbamoylase with the amino acid sequence shown as SEQ ID NO.2 into tryptophan (E138W); or replacing aspartic acid at position 187 with asparagine (D187N); or replacement of alanine at position 200 with serine (a 200S); or replacing alanine at position 200 with asparagine (a 200N); or alanine at position 200 with glutamic acid (a 200E); or replacing serine 207 with alanine (S207A); or arginine at position 211 is replaced by glycine (R211G).

Furthermore, the mutant is obtained by replacing arginine at the 211 th site of D-carbamyl hydrolase with glycine, wherein the amino acid sequence of the D-carbamyl hydrolase is shown as SEQ ID NO.2, and simultaneously mutating glutamic acid at the 138 th site to tryptophan (E138W/R211G); or simultaneously replacing alanine at position 200 with serine (A200S/R211G); or simultaneously replacing alanine at position 200 with asparagine (A200N/R211G); or simultaneously replacing alanine at position 200 with glutamic acid (A200E/R211G); or simultaneously replacing serine at position 207 with alanine (S207A/R211G).

Further, the mutant is obtained by replacing arginine at position 211 of D-carbamoylase with glycine, alanine at position 200 with asparagine, and simultaneously replacing aspartic acid at position 187 with asparagine (R211G/A200N/D187N), wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 2; or arginine at position 211 is replaced by glycine, glutamic acid at position 138 is replaced by tryptophan, and serine at position 207 is replaced by alanine (R211G/E138W/S207A); or arginine at position 211 is replaced by glycine, alanine at position 200 is replaced by asparagine, and serine at position 207 is replaced by alanine (R211G/A200N/S207A); or replacing arginine at the 211 position with glycine, simultaneously replacing serine at the 207 position with alanine, and replacing aspartic acid at the 187 position with asparagine (R211G/S207A/D187N).

Further, the mutant is obtained by replacing arginine at position 211 of D-carbamoylase with glycine, alanine at position 200 with asparagine, aspartic acid at position 187 with asparagine, and serine at position 207 with alanine, wherein the amino acid sequence of the mutant is shown as SEQ ID NO.2 (R211G/A200N/D187N/S207A); or arginine at the 211 position is replaced by glycine, alanine at the 200 position is replaced by asparagine, aspartic acid at the 187 position is replaced by asparagine, and glutamic acid at the 138 position is replaced by tryptophan (R211G/A200N/D187N/E138W).

Further, the mutant is obtained by replacing arginine at position 211 of D-carbamoylase with glycine, alanine at position 200 with asparagine, aspartic acid at position 187 with asparagine, serine at position 207 with alanine, and glutamic acid at position 138 with tryptophan (R211G/A200N/D187N/S207A/E138W), wherein the amino acid sequence of the D-carbamoylase is shown as SEQ ID NO. 2.

It is a second object of the present invention to provide a gene encoding the mutant.

The third purpose of the invention is to provide an expression vector carrying the gene.

The fourth purpose of the invention is to provide a recombinant bacterium for expressing the mutant.

Further, the construction method of the recombinant bacterium comprises the following steps: cloning the nucleic acid molecule encoding the mutant into a recombinant vector, and transforming the obtained recombinant vector into host bacteria to obtain the recombinant bacteria.

Further, the host of the recombinant bacterium is Escherichia coli (Escherichia coli), and the plasmid is pET28a (+).

Further, the host of the recombinant bacterium is e.coli BL21(DE 3).

The fifth purpose of the invention is to provide the method for producing the D-carbamoylase by the recombinant strain, which comprises the following specific steps: inoculating the recombinant bacteria into LB culture medium containing 40-60 mug/mL kanamycin sulfate, carrying out shake culture at 30-40 ℃ at 100-200 rpm, and carrying out OD (absorbance) of culture solution ₆₀₀ When the concentration reaches 0.5-1.0, adding 0.05-1.0 mM IPTG for induction at the induction temperature of 16-30 ℃ for 5-12 h to obtain the recombinant D-carbamyl hydrolase.

The sixth object of the present invention is to provide the use of the D-carbamoyl hydrolase mutant for preparing an optically pure D-amino acid.

Furthermore, the D-carbamyl hydrolase mutant is used as a catalyst to catalyze a substrate to generate D-amino acid, wherein the substrate is DL-N-carbamyl tryptophan, DL-N-carbamyl phenylalanine, DL-N-carbamyl phenylglycine, DL-N-carbamyl methionine, DL-N-carbamyl tryptophan, DL-N-carbamyl o-chlorophenylglycine, DL-N-carbamyl leucine or DL-N-carbamyl isoleucine.

Further, the application specifically comprises the following steps: constructing a reaction system, wherein the concentration of L-indolylmethyl is 10 mM-1M, the dosage of the D-carbamoylase mutant is 1-10 kU/L, and the concentration of a phosphate buffer solution is 0.05-0.15M; reacting for 1-24 h at the temperature of 30-35 ℃ and under the condition of pH 6-8.

The invention has the beneficial effects that:

the D-carbamyl hydrolase mutation has high activity on various N-carbamyl amino acids, and can catalyze various aliphatic or aryl substituted amino acid substrates, particularly D-N-carbamyl amino acid substrates with high steric hindrance. The mutants are used for the validation that 500mM L-indolylmethylhydantoin is catalyzed by a hydantoinase process cascade reaction, wherein the yield of a product of the five mutants A200N/R211G/D187N/S207A/E138W in 12 hours of reaction reaches 81.7 percent, compared with WT (40.1 percent), the yield of the product is improved by 2 times, and the reaction time is greatly shortened and only needs 12 hours. The D-carbamyl hydrolase mutant obtained by the invention is particularly suitable for preparing optically pure D-amino acid by a hydantoinase process cascade reaction, and has good industrial application prospect.

Drawings

FIG. 1 is a diagram of a whole plasmid PCR nucleic acid electrophoresis of a D-carbamoyl hydrolase mutant;

FIG. 2 is an SDS-PAGE electrophoresis of the expression and purification of the five most preferred mutants of D-carbamoylase; lane 1: crude enzyme solution, 2: marker; 3: pure enzyme

FIG. 3 is a chiral liquid chromatogram of D-carbamoylase mutant catalyzing D-N-carbamoyltryptophan;

FIG. 4 is a chiral liquid chromatogram of D-N-carbamoyl p-hydroxyphenylalanine catalyzed by D-carbamoyl hydrolase mutant.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The enzymatic activity of the D-N-carbamoylhydrolase on the substrate DL-N-carbamoyltryptophan was determined. The measuring system is as follows: appropriate amount of enzyme solution, 10 mmol. L ^-1 DL-N-carbamoyltryptophan. Standing and reacting for 10min at 30 ℃. Reaction ofAnd after the end, sampling and carrying out liquid phase detection. Liquid phase detection conditions: the column was Diamonsil Plus C18(25cm × 4.6mm,5 μm), the mobile phase was acetonitrile: potassium dihydrogen phosphate (25: 75) at a flow rate of 0.2 to 1mL/min ^-1 The detection wavelength is 210 nm.

Definition of enzyme activity unit (U):

the amount of enzyme required by the D-N-carbamoylhydrolase to catalyze the formation of 1. mu. mol of D-tryptophan from the substrate DL-N-carbamoyltryptophan at 30 ℃ is defined as one enzyme activity unit (U)

Example 1: construction of D-carbamoylase mutant Gene and recombinant expression transformant

The amino acid residues of Phe53, Pro130, His143, Glu145, Asn172, Arg174, Arg175, Thr195, Asn196, Thr197, Pro198, Ala200, Asp201, Ser204 and Arg211 are subjected to site-directed saturation mutagenesis by a whole-plasmid PCR method, the primer design is shown in Table 1 (all described in the 5 '-3' direction), and the mutation sites are underlined.

TABLE 1 design table of site-directed saturation mutagenesis primers

Site-directed mutagenesis was performed on the amino acid residues of Ala49, Arg61, Tyr67, Ser92, Glu138, Arg139, His141, Ala169, Val177, Asp187, Leu192, Ser202, Leu203, Gly205, Ser207, Met212, Ser227, Thr301 by a whole-plasmid PCR method, with primer design as shown in table 2 (all described in 5 '-3' direction), and underlined representing the mutation sites:

TABLE 2 design of site-directed mutagenesis primers

The PCR reaction system is as follows: the PCR reaction system (50. mu.L) included l.0. mu.L of KOD enzyme (2.5U/mL), l.0. mu.L of template (5-50ng), 4.0. mu.L of dNTP, 5.0. mu.L of 10 × reaction buffer, 1.0. mu.L of each of the upstream and downstream primers, and ddH ₂ O make up to 50. mu.L.

The PCR amplification procedure was: (1) denaturation at 94 ℃ for 3min, (2) denaturation at 94 ℃ for 30sec, (3) annealing at 54 ℃ for 30sec, (4) extension at 72 ℃ for 150sec, repeating steps (2) - (4) for 10-15 cycles, finally extension at 72 ℃ for 10min, and storing the PCR amplification product at 4 ℃.

After the PCR was completed, DpnI restriction enzyme was added to the reaction mixture and incubated at 37 ℃ for 1h, followed by CaCl ₂ Thermal transformation method 10. mu.L of digested PCR reaction solution was transferred into 50. mu.L of E.coli BL21(DE3) competent cells, and uniformly spread on LB agar plate containing 50. mu.g/ml kanamycin sulfate, and cultured in inversion at 37 ℃ for 12 hours.

Example 2: expression and purification of D-carbamyl hydrolase and its mutant

The recombinant E.coli containing the mutant plasmid was inoculated in an amount of 2% of the transfer amount to LB medium containing kanamycin sulfate (50. mu.g/mL), cultured with shaking at 37 ℃ and 200rpm, and the absorbance OD of the culture solution was adjusted ₆₀₀ When the concentration reaches 0.8, 0.2mM IPTG is added for induction at the induction temperature of 25 ℃, after 12 hours of induction, the thalli of the high-efficiency expression recombinant D-carbamoylase mutant are obtained by centrifugation at 8000rpm for 5min, the collected thalli are suspended in Tris-HCl buffer solution (100mM, pH 8.0) and are crushed by ultrasound.

The column used for purification was HisTrap HP 5mL, a nickel affinity column, and affinity chromatography was performed using a histidine tag on the recombinant protein. The recombinant D-carbamoylase mutant is obtained by firstly equilibrating the nickel column with solution A, loading the crude enzyme solution, eluting the breakthrough peak with solution A (25mM Tris, 500mM NaCl,20mM imidazole, pH 7.4), after equilibration, carrying out gradient elution with solution B (25mM Tris, 500mM NaCl, 500mM imidazole, pH 7.4), and eluting the recombinant protein bound to the nickel column. The purified protein was subjected to activity determination and SDS-PAGE analysis. After nickel column purification, a single band is shown at about 38kDa, and the impurity protein is less, which indicates that the column purification effect is better. The purified D-carbamoylase protein was then replaced into Tris-HCl (l00mM, pH 8.0) buffer using a His Trap desaling Desalting column (GE Healthcare).

Example 3: kinetic parameters and cascade conversion analysis of D-carbamoyl hydrolase mutant

And (3) determining the kinetic parameters of the NiHyuC and the mutant on the substrate DL-N-carbamoyl-tryptophan. The kinetic parameter measurement system is listed below: Tris-HCl buffer (100 mmol. L) ^-1 pH 8.0), DL-N-carbamoyl-tryptophan (0 to 20 mmol. multidot.L) ^-1 ). The reaction rate was characterized by calculating the specific enzyme activity, and thus the kinetic parameters were calculated.

Since there are few reports on the modification of the enzymatic activity of carbamoylase and no mutation site for reference, the present patent uses EasyModeller to perform homologous modeling using D-carbamoylase (PDB No.: 1fo6) from Agrobacterium radiobacter as a template, and then performs model verification and evaluation.

Docking the substrate D-N-carbamoyltryptophan to the model protein structure using molecular docking after obtaining the structure of the protein, followed by selection of the substrate

Sites within the range were subjected to construction and screening of a library of saturation mutations. The mutation sites involved are: performing shake flask rescreening on excellent mutants obtained by primary screening of Phe53, Pro130, His143, Glu145, Asn172, Arg174, Arg175, Thr195, Asn196, Thr197, Pro198, Ala200, Asp201, Ser204 and Arg211, and performing protein purification and kinetic parameter determination on mutants with improved rescreening activity. As shown in Table 3, six mutants of R211G, R211S, A200E, A200N, A200S and A200H are obtained by screening, wherein the mutants with remarkably improved kinetic parameters are respectively R211G and k thereof _cat /K _m Is 87.0min ^-1· mM ^-1 Is WT (k) _cat /K _m It is 25.7min ^-1· mM ^-1 ) 3.4 times of; A200E, k thereof _cat /K _m It is 96.4min ^-1· mM ^-1 3.8 times of WT; A200N, k thereof _cat /K _m Is 88.0min ^-1· mM ^-1 3.4 times of WT; A200S, k thereof _cat /K _m Is 109min ^-1· mM ^-1 4.2 times of WT; A200H, k thereof _cat /K _m It is 67.7min ^-1· mM ^-1 2.6 times of WT. Then to k _cat /K _m Compared with WT improved single-point mutants, the verification of conversion of 200mM L-indolylhydantoine catalyzed by cascade reaction is carried out, the enzyme addition amounts of the three enzymes are respectively 15kU/L AaHyuA,20kU/L AtHyuH and 5kU/L NiHyuC, the yield of the products is respectively 97.7%, 97.8%, 92.7%, 94.2% and 85.6%, and the yield of WT is 79.3%.

Then according to the evolutionary conservative analysis of the multiple sequence alignment result of the amino acid sequence of the family, Ala49, Arg61, Tyr67, Ser92, Glu138, Arg139, His141, Ala169, Val177, Asp187, Leu192, Ser202, Leu203, Gly205, Ser207, Met212, Ser227 and Thr301 sites are selected to carry out corresponding site-directed mutagenesis, the activity of the crude enzyme is determined, and the mutant with the activity of the crude enzyme improved compared with that of WT is subjected to protein purification and kinetic parameter determination. The results are shown in Table 3: wherein k is compared to WT _cat /K _m The mutants with significant improvement are R61Q, Y67F, E138W, E138Q, R139A, A169C, D187N, L192M, S202A, G205P, S207T, S207A, S227A, M212W, wherein k of R61Q _cat /K _m Is 106min ^-1· mM ^-1 4.1 times of WT; k of Y67F _cat /K _m Is 93.3min ^-1· mM ^-1 3.6 times of WT; k of E138W _cat /K _m Is 113min ^-1· mM ^-1 4.4 times of WT; k of E138Q _cat /K _m Is 68.7min ^-1· mM ^-1 2.7 times of WT; k of R139A _cat /K _m It is 70.5min ^-1· mM ^-1 2.7 times of WT; k of A169C _cat /K _m It is 91.3min ^-1· mM ^-1 3.6 times of WT; k of D187N _cat /K _m Is 121min ^-1· mM ^-1 4.7 times of WT; k of L192M _cat /K _m Is 130min ^-1· mM ^-1 5.1 times of WT; k of S202A _cat /K _m It is 82.8min ^-1· mM ^-1 3.2 times of WT; k of G205P _cat /K _m It is 55.8min ^-1· mM ^-1 2.2 times of WT; k of S207T _cat /K _m Is 123min ^-1· mM ^-1 4.8 times of WT; k of S207A _cat /K _m Is 62.3min ^-1· mM ^-1 2.4 times of WT; k of M212W _cat /K _m It is 79.8min ^-1· mM ^-1 3.1 times of WT; k of S227A _cat /K _m Is 81.1min ^-1· mM ^-1 3.2 times of WT.

Then the same pair of k _cat /K _m Compared with the mutant with the improved quality of WT, the mutant is verified to be converted by catalyzing 200mM L-indolylhydantoine through cascade reaction, the enzyme addition amounts of the three enzymes are respectively 15kU/L AaHyuA,20kU/LAtHyuH and 5kU/L NiHyuC, and the product yield of the mutants is higher than that of WT (79.3 percent) respectively: Y67F 89.2.2%, E138W 93.8.8%, A169C 82.7.7%, D187N 94.9.9%, S202A 82.8.8%, G205P 89.5.5%, S207A 92.4.4%, S227A 89.3.3%, less than or equal to WT, respectively: R61Q 77.1.1%, E138Q 74.2.2%, R139A 79%, L192M 72.1.1%, S207T 77.7.7%, M212W 79.3.3%. And then discarding mutants with unobvious cascade reaction transformation effects, and finally selecting single-point mutants (R211G, A200E, A200N, A200S, Y67F, E138W, D187N, G205P, S207A and S227A) with the product yield higher than WT 10% to perform subsequent random combination mutation.

TABLE 3 demonstration of kinetic parameters of single-point mutants of D-carbamoylase and catalytic 200mM substrate cascade

Constructing and screening the selected single-point mutants in a random combination mutation library, carrying out subsequent shake flask culture and secondary screening on the obtained multiple mutants, carrying out protein purification on the multiple mutants with higher enzyme activity than the single-point mutants, and measuring dynamic parameters, wherein the results are shown in Table 4, the catalytic efficiency of the obtained double mutants A200S/R211G, A200N/R211G, A200E/R211G, E138W/R211G, D187N/R211G and S207A/R211G are greatly improved compared with the single-point mutants and WT, and k 207A/R211G is greatly improved, and the K is K _cat /K _m Are respectively 160min ^-1· mM ^-1 ，246min ^-1· mM ^-1 ，191min ^-1· mM ^-1 ，286min ^-1· mM ^-1 ，193min ^-1· mM ^-1 ，275min ^-1· mM ^-1 6.2 times, 9.6 times, 7.4 times, 11.1 times, 7.5 times and 10.7 times of WT respectively, wherein three mutants in the screening result comprise A200N/R211G/D187N, A200N/R211G/E138W, A200N/R211G/S207A, E138W/R211W/S207W, E138W/R211W/D187W, S207W/R211W/D187W, wherein compared with double mutation and WT catalytic efficiency, the three mutants only comprise A200W/R211/D187W, A200W/R211W/S207W, E138W/R W/S36207, S207W/R211/D187/D W and k is higher than that of the double mutation and WT catalytic efficiency are obviously improved _cat /K _m Are respectively 631min ^-1· mM ^-1 ，808min ^-1· mM ^-1 ，600min ^-1· mM ^-1 ，638min ^-1· mM ^-1 Respectively 24.6 times, 31.4 times, 23.3 times and 24.8 times of WT. Four mutants obtained by screening comprise A200N/R211G/D187N/S207A, A200N/R211G/D187N/E138W, A200N/R211G/S207A/E138W, and k of the four mutants _cat /K _m Compared with WT and triple mutation, the improvement is only obvious A200N/R211G/D187N/S207A, and is 1135min ^-1· mM ^-1 The optimal mutant obtained by screening is five mutants, namely A200N/R211G/D187N/S207A/E138W, and k of the five mutants is 44.2 times of WT _cat /K _m Is 1820min ^-1· mM ^-1 70.8 times of WT. Also in order to verify the transformation effect of these multiple mutants, we performed a test for verifying the transformation effect of the cascade reaction. Since the previous single-point mutants R211G and A200E had completed the complete conversion of 200mM L-indolylhydantoine, we raised the substrate L-indolylhydantoine concentration to 500mM when performing the validation experiment of the multiple mutants, with the enzyme addition amounts of the three enzymes being 15kU/L AaHyuA,20kU/L AtHyuH,5kU/L NiHyuC, respectively. The results are shown in Table 4, wherein the product yield of the five-mutant A200N/R211G/D187N/S207A/E138W reaches 81.7% in 12h reaction, compared with WT (40.1%), the product yield is improved by 2 times, and the reaction time is greatly shortened and only needs 12 h. For other multi-mutants, the product yield of the four-mutant A200N/R211G/D187N/S207A in 24h is 79.3%, and the product yield of the three-mutant A200N/R211G/D187N, A200N/R211G/S207A, E138W/R211G/S207A and S207A/R211G/D187N in 24h are 76.4%, 79.8%, 75.3%, 78.4%, 79.8% respectively, 75.3% and 78.4% respectivelyThe product yields of the double mutants A200S/R211G, A200N/R211G, A200E/R211G, E138W/R211G, D187N/R211G and S207A/R211G are 67.2%, 65.0%, 65.7%, 70.7% and 66.4%, respectively, and are all improved to a greater extent than that of WT.

TABLE 4 kinetic parameters of D-carbamoylase combinatorial mutants and validation of catalytic 500mM substrate cascade

Example 4: time course of use of D-carbamoylase mutant for preparing D-tryptophan by cascade reaction

Taking 5kU/L of the obtained recombinant D-N-carbamoylase (wild type), 15kU/L of hydantoin racemase (AaHyuA) and 20kU/L D-hydantoin enzyme (AtHyuH) in Tris-HCl buffer solution (pH 6-8, 100 mmol.L ^-1 ) In the solution, 20% PEG400, 500 mmol. L are added ^-1 L-indolylhydrogen, the total volume of the reaction solution is 10 mL. The reaction was placed at 30 ℃ and samples were taken to examine the conversion process under the following conditions: diamonsil Plus C18 column (25cm × 4.6mm,5 μm), detection wavelength 210nm, mobile phase acetonitrile: monopotassium phosphate (10-30: 90-70) with the flow rate of 0.5-1 mL/min.

Meanwhile, 6 reactions of mutants R211G, E138W/R211G, A200N/R211G/D187N, A200N/R211G/D187N/S207A, A200N/R211G/S207A/D187N/E138W and a substrate L-indolylmethylhydantoin are established by the same method. The reaction was carried out at 30 ℃ and 200rpm for 24h, with a constant pH of 8.0. The time course of the reaction process is shown in tables 5 and 6, and it can be seen from the tables that the wild type carbamoyl hydrolase is slow in reaction, the final substrate conversion rate is 49.4% at 24h (table 5), the yield is 40% (table 6), while the mutant R211G has exceeded 60% at 6h (table 5), the conversion rate slowly increases at the later stage of the reaction, and reaches 70% at 24h, the product yield is 55.8% (table 6); compared with the single mutation R211G, the reaction rate of the double mutant E138W/R211G is increased, the conversion rate of the substrate reaches 61.4% after 3 hours of reaction, the product yield is 54.3%, the conversion rate of the substrate reaches 85.8% after 24 hours, and the product yield is 70.6%; the reaction rate of the triple mutant A200N/R211G/D187N is further improved compared with that of the double mutant, the substrate conversion rate reaches 88.5% at the reaction time of 3h, the product yield is 75.1%, the substrate conversion rate reaches 94.7% at 24h and the product yield is 76.4% as the reaction time goes on, the reaction rate of the triple mutant is reduced in the four mutants A200N/R211G/D187N/S207A, but the substrate conversion rate at the final 24h can reach 94.5% and the product yield is 79.2%, the five mutant A200N/R211G/D187N/S207A/E138W has no further improvement on the final substrate conversion rate compared with the four mutant, is 94.5% and the product yield is 81.7%, but the initial reaction rate is improved by 2 times compared with the four mutant, the substrate conversion rate is improved from 14.2% (four mutants) to 25.2% (five mutants) at the reaction time of 5min, and the reaction time can be known, compared with WT, the single mutant or the multiple mutants have obvious improvement on the conversion rate and the reaction rate, and the loading amount of enzyme is greatly reduced when catalyzing the conversion of 500mM substrate, and is only 15kU/L AaHyuA,20kU/L AtHyuH and 5 kU/LNiHyuC.

TABLE 5 progress of the conversion of wild type and different mutants to catalyze 500mM L-indolylhydhydantoin

Conversion/％	5min	3h	6h	12h	24h
						WT	2.3	31.7	39.9	46.5	49.4
R211G	5.4	53.3	58.8	62.5	67.3
						E138W/R211G	4.9	61.4	64.2	73.0	85.8
A200N/R211G/D187N	16.7	88.5	88.5	91.9	94.7
						A200N/R211G/D187N/S207A	14.2	84.5	94.6	94.3	94.5
A200N/R211G/D187N/S207A/E138W	25.2	88.0	93.5	93.5	94.5

TABLE 6 progress of the product yield of 500mM L-indolylhydrogen catalysed by the wild type and different mutants

Yield/％	5min	3h	6h	12h	24h
						WT	1.0	26.9	35.0	40.2	40.2
R211G	3.6	46.6	52.0	54.7	55.8
						E138W/R211G	4.0	54.3	57.0	63.8	65.7
A200N/R211G/D187N	15.9	75.1	75.6	76.3	76.4
						A200N/R211G/D187N/S207A	13.5	76.4	78.8	79.1	79.3
A200N/R211G/D187N/S207A/E138W	23.5	79.2	79.4	80.6	81.7

Example 5: application of D-carbamyl hydrolase mutant in time course of preparing D-p-hydroxyphenylalanine by cascade reaction

Taking 10kU/L of the obtained recombinant D-N-carbamyl hydrolase (wild type), 15kU/L of hydantoin racemase (AaHyuA) and 20kU/L D-hydantoin enzyme (AtHyuH) in Tris-HCl buffer solution (pH 6-8, 100 mmol.L ^-1 ) Adding 5-10% PEG400, 500 mmol. L ^-1 The total volume of the reaction solution is 10 mL. The reaction is kept at 30 DEG CSampling and detecting the transformation process under the following conditions: diamonsil Plus C18 column (25cm × 4.6mm,5 μm), detection wavelength 210nm, mobile phase acetonitrile: monopotassium phosphate (10-30: 90-70) with the flow rate of 0.5-1 mL/min.

Meanwhile, 6 mutants of R211G, E138W/R211G, A200N/R211G/D187N, A200N/R211G/D187N/S207A, A200N/R211G/S207A/D187N/E138W and a substrate L-p-hydroxyhydantoin are established by the same method to react. The reaction was carried out at 30 ℃ and 200rpm for 24h, with a constant pH of 8.0. The time course of the reaction process is shown in tables 7 and 8, and the table shows that the wild type carbamyl hydrolase is slow in reaction, the final substrate conversion rate is 49.4 percent (table 7) and the yield is 33.2 percent (table 8) at 24h, while the conversion rate of the mutant R211G is 55.8 percent (table 7) at 6h, the conversion rate slowly rises at the later stage of the reaction, 63.3 percent at 24h and the product yield is 43.8 percent (table 8); compared with the single mutation R211G, the reaction rate of the double mutant E138W/R211G is increased, the conversion rate of the substrate reaches 61.1% after 3 hours of reaction, the product yield is 41.1%, the conversion rate of the substrate reaches 75.8% after 24 hours, and the product yield is 51.7%; compared with the double mutant, the reaction rate of the three mutants A200N/R211G/D187N is further improved, the substrate conversion rate reaches 71.5% and the product yield reaches 65.1% in the reaction time of 3h, the substrate conversion rate reaches 83.7% and the product yield reaches 66.4% in 24h along with the progress of the reaction time, the substrate conversion rate in the final 24h of the four mutants A200N/R211G/D187/S187N/S207A can reach 89.5% and the product yield reaches 79.1%, compared with the four mutants, the five mutants A200N/R211G/D187N/S207A/E138W are further improved in the final substrate conversion rate to 94.1% and the product yield to 81.0%, and in conclusion, compared with the single mutant or the multiple mutants, the conversion rate and the reaction rate are both obviously improved, the loading amount of the enzyme is greatly reduced and is only 15 kU/LAaWT when 500mM substrate conversion is catalyzed, 20kU/L AtHyuH,10kU/L NiHyuC.

TABLE 7 progress of the conversion of wild type and different mutants catalysing 500mM L-p-hydroxyphenylhydantoin

Conversion/％	5min	3h	6h	12h	24h
						WT	1.3	31.2	35.9	46.2	49.4
R211G	3.4	50.3	55.8	60.5	63.3
						E138W/R211G	4.2	61.1	64.2	71.1	75.8
A200N/R211G/D187N	15.7	71.5	78.5	81.9	83.7
						A200N/R211G/D187N/S207A	18.3	80.5	84.6	87.3	89.5
A200N/R211G/D187N/S207A/E138W	21.2	82.1	91.5	93.1	94.1

TABLE 8 Process for the product yield of 500mM L-p-hydroxyphenylhydantoin catalyzed by wild type and different mutants

Yield/％	5min	3h	6h	12h	24h
						WT	1.0	22.9	31.1	33.2	33.2
R211G	2.6	34.6	42.2	43.7	43.8
						E138W/R211G	4.0	41.1	47.0	50.8	51.7
A200N/R211G/D187N	12.9	65.1	65.6	66.3	66.4
						A200N/R211G/D187N/S207A	13.1	70.4	78.8	79.0	79.1
A200N/R211G/D187N/S207A/E138W	19.5	79.0	79.4	80.1	81.0

Therefore, the D-carbamoylase mutant has very good performance in the aspects of high-efficiency and high-stereoselectivity catalysis of D-carbamoyltryptophan, and has higher catalytic efficiency and high stereoselectivity for other aromatic D-N-carbamoylamino acid substrates.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of south of the Yangtze river

<120> D-carbamoyl hydrolase mutant and application thereof in D-aromatic amino acid synthesis

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Met Thr Arg Arg Ile Arg Ile Gly Gly Ala Gln Met Gly Ala Ile Ser

1 5 10 15

Arg Ser Asp Ser Lys Lys Glu Ile Val Asp Arg Leu Ile Ala Leu Leu

20 25 30

Arg Gln Ala Ser Glu Lys Gly Cys Glu Leu Val Val Phe Pro Glu Leu

35 40 45

Ala Leu Ser Thr Phe Phe Pro Arg Trp Tyr Ala Glu Arg Asp Gly Met

50 55 60

Asp Gly Tyr Phe Glu Asp Gly Met Pro Asn Ala Ala Thr Leu Pro Leu

65 70 75 80

Phe Glu Glu Ala Arg Arg Leu Gly Ile Gly Phe Ser Leu Gly Tyr Ala

85 90 95

Glu Leu Val Gln Glu Asp Gly Arg Val Arg Arg Phe Asn Thr Thr Val

100 105 110

Leu Val Glu Arg Asn Gly Glu Ile Val Gly Lys Tyr Arg Lys Ile His

115 120 125

Leu Pro Gly His Ala Glu Tyr Glu Pro Glu Arg Ser His Gln His Leu

130 135 140

Glu Lys Arg Tyr Phe Glu Val Gly Asn Thr Gly Phe Gln Val Trp Asp

145 150 155 160

Ala Phe Gly Gly Arg Val Gly Met Ala Ile Cys Asn Asp Arg Arg Trp

165 170 175

Val Glu Thr Tyr Arg Val Met Gly Leu Gln Asp Val Glu Leu Ile Leu

180 185 190

Ile Gly Tyr Asn Thr Pro Val Ala Asp Ser Leu Ser Gly Glu Ser Glu

195 200 205

Thr Leu Arg Met Phe His Asn His Leu Thr Met Gln Ala Gly Ala Tyr

210 215 220

Gln Asn Ser Thr Trp Val Val Gly Val Ala Lys Ala Gly Val Glu Asp

225 230 235 240

Gly His Arg Leu Met Gly Gly Ser Val Ile Val Ala Pro Thr Gly Glu

245 250 255

Ile Val Ala Gln Ala Met Thr Glu Gly Asp Glu Leu Ile Val Ala Asp

260 265 270

Cys Asp Leu Asp Arg Cys Arg Tyr Tyr Lys Ser His Ile Phe Asn Phe

275 280 285

Ala Ala His Arg Arg Pro Glu Phe Tyr Gln Arg Ile Thr Ser Gln Thr

290 295 300

Gly Val Glu

305

Claims (5)

1. A D-carbamoyl hydrolase mutant characterized in that the 211 th arginine of D-carbamoyl hydrolase having an amino acid sequence shown in SEQ ID NO.2 is replaced with glycine; or

Replacing arginine at the 211 th site with glycine, and mutating glutamic acid at the 138 th site into tryptophan; or arginine at position 211 is replaced by glycine, and alanine at position 200 is replaced by serine; or arginine at position 211 is replaced by glycine, and alanine at position 200 is replaced by asparagine; or arginine at position 211 is replaced by glycine, and alanine at position 200 is replaced by glutamic acid; or arginine at position 211 is replaced by glycine, and serine at position 207 is replaced by alanine; or

Replacing arginine at position 211 with glycine, alanine at position 200 with asparagine, and aspartic acid at position 187 with asparagine; or replacing arginine at position 211 with glycine, simultaneously replacing glutamic acid at position 138 with tryptophan, and replacing serine at position 207 with alanine; or arginine at position 211 is replaced by glycine, alanine at position 200 is replaced by asparagine, and serine at position 207 is replaced by alanine; or replacing arginine at position 211 with glycine, serine at position 207 with alanine, and aspartic acid at position 187 with asparagine; or

Replacing arginine at position 211 with glycine, alanine at position 200 with asparagine, aspartic acid at position 187 with asparagine, and serine at position 207 with alanine; or arginine at position 211 is replaced by glycine, alanine at position 200 is replaced by asparagine, aspartic acid at position 187 is replaced by asparagine, and glutamic acid at position 138 is replaced by tryptophan; or

Arginine at position 211 was replaced with glycine, alanine at position 200 with asparagine, aspartic acid at position 187 with asparagine, serine at position 207 with alanine, and glutamic acid at position 138 with tryptophan.

2. A gene encoding the D-carbamoyl hydrolase mutant according to claim 1.

3. An expression vector carrying the gene of claim 2.

4. A recombinant bacterium expressing the D-carbamoyl hydrolase mutant according to claim 1.

5. Use of a D-carbamoylase mutant according to claim 1 for the preparation of an optically pure D-amino acid.

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